The concentration dependence of the hemoglobin mutual diffusion coefficient

Laser correlation Spectroscopy was used to measure the mutual diffusion coefficient, D, of human cyanomethemoglobin (Fe⁺⁺⁺:CN) at varying protein concentrations. These measurements were male at 20°C in a 0.1 M phosphate buffer solution at pH 7.0. For low protein concentrations we find D = (6.43 ± 0.26) × 10^?7 cm²/S and that there is a near linear decrease from this value at higher concentrations. The linear relation between the diffusion coefficient and protein concentration allows us to deduce the value of the linear frictional volume fraction coefficient, K_f= 7.75. and to extrapolate to hemoglobin concentrations equivalent to that in the red blood cell where we estimate D = 4.25 × 10^?7 cm²/s Various theoretical predictions of the dependence of the mutual diffusion coefficient on concentration are tested; we find that the generalized Stokes-Einstein relation can be made to fit our high concentration data if we assume a hard-sphere model and if we include a term involving a hydrodynamic interaction integral.     

Pulsed field gradient nmr determination of the temperature dependence of the tracer diffusion coefficient of hemoglobin

We have studied the temperature dependence of the tracer diffusion coefficient of carbonmonoxy hemoglobin A (HbA-CO) by means of pulsed-field gradient nmr (PFG-nmr). Measurements were made over the temperature range from 15 to 35°C for samples having concentrations 7.4 and 16.7 g/dL. No significant deviations were found from the predictions of the Stokes-Einstein relation. Thus, this work does not corroborate the recently proposed conformational change in hemoglobin at 22°C. The advantages of PFG-nmr for the study of hemoglobin are discussed.     

Post-harvest Changes in the Respiratory Activity of and Ethanol Accumulation in Fresh Rice Grains

Fresh rice grains stored under anaerobic conditions at 4°C showed a strong activity of anaerobic respiration at 30°C. When stored at 30°C, the rates of both oxygen consumption and carbon dioxide evolution declined rapidly. The ethanol content in paddy at the post-harvest stage was increased at 4°C, whereas no significant accumulation of ethanol was observed at 30°C. The accumulated ethanol in paddy was depleted as the storage temperature was raised from 4 to 30°C. In contrast, a temperature-dependent accumulation was observed with a lowering from 30 to 4°C. On the other hand, ethanol content in brown rice changed little with storage temperature. On the basis of these results, it is assumed that ethanol is more easily accumulated in the rice grains against diffusion to the atmosphere at the lower temperature.     

Dynamic Light Scattering Studies on Hydrodynamic Properties of Fibrinogen-Fibronectin Complex

Abstract

A high molecular weight ‘cryogel’ was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37°C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20°C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20°C in 0.05 M Tris- HCl(pH7.4) containing 0.5 M NaCl was estimated as 8.5× 10^?8 cm²s^?1. The complex did not dissociate over the temperature range from 20 to 37°C. The diffusion coefficients of the complex decreased significantly at 12°C and 40°C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40°C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15°C, but not at near- freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.     

Testing the drivers of the temperature–size covariance using artificial selection

Body size often declines with increasing temperature. Although there is ample evidence for this effect to be adaptive, it remains unclear whether size shrinking at warmer temperatures is driven by specific properties of being smaller (e.g., surface to volume ratio) or by traits that are correlated with size (e.g., metabolism, growth). We used 290 generations (22 months) of artificial selection on a unicellular phytoplankton species to evolve a 13-fold difference in volume between small-selected and large-selected cells and tested their performance at 22°C (usual temperature), 18°C (−4), and 26°C (+4). Warmer temperatures increased fitness in small-selected individuals and reduced fitness in large-selected ones, indicating changes in size alone are sufficient to mediate temperature-dependent performance. Our results are incompatible with the often-cited geometric argument of warmer temperature intensifying resource limitation. Instead, we find evidence that is consistent with larger cells being more vulnerable to reactive oxygen species. By engineering cells of different sizes, our results suggest that smaller-celled species are pre-adapted for higher temperatures. We discuss the potential repercussions for global carbon cycles and the biological pump under climate warming.     

Intensity-fluctuation spectroscopy and tRNA conformation. II. Changes of size and shape of tRNA in the melting process

We have measured the translational (D^T) and rotational (D^R) diffusion coefficients of bulk tRNA from baker's yeast during the thermal unfolding process by means of photon-correlation spectroscopy. It should be noted that our estimate of the rotational diffusion coefficient represented, for the first time, measurements on a small macromolecule in solution by the photoelectron time-of-arrival technique with a delay-time resolution of 1 nsec. The melting curves expressed in terms of δD^T vs temperature were consistent with the literature data in revealing the melting steps and their dependence on NaCl concentration. Additionally, it was possible to prove the existence of an intermediate, more compact structure during the initial steps of the thermal unfolding process. We found that the temperature ranges over which this intermediate structure appears depend strongly on salt concentration. By utilizing both translational and rotational diffusion coefficients and Perrin's equations for ellipsoids of revolution, we have computed the values of the equivalent length and width of tRNA molecules in solution at four different temperatures for NaCl concentrations of 0.2, 0.5, and 1M. The approximate model of ellipsoids of revolution also permits us to obtain an estimate of the radius of gyration, which is in very good agreement with literature data measured by means of small-angle x-ray scattering. Furthermore, we have measured the shape and size changes of tRNA with varying NaCl concentrations at room temperatures (25°C). The molecule becomes smaller and more spherical when NaCl concentration increases. As a result of partial melting at 70°C, the macromolecule is surprisingly elongated with an approximate axial ratio of 8:1 and has dimensions of about 180/22Å. Such information on conformational changes by a simultaneous determination of rotational and translational diffusion coefficients illustrates the potential of this approach, not available by other methods.     

Self-association of β-lactoglobulin A in acid solution. I. Translational diffusion coefficients

We report measurements of the diffusion coefficient of β-lactoglobulin A (βLG-A) at pH = 5.60 and 4.58 in 0.10 ionic strength acetate buffer by the techniques of analog photocurrent signal correlation and digital single-clipped photon correlation. At a concentration of 21 mg/ml and a pH of 4.58, the self-association of β-lactoglobulin can be represented by a simple dimer–octamer equilibrium model. We determined the translational diffusion coefficient of the dimer and that of the octamer using the scattering results of Kumosinski and Timasheff in a dimer–octamer mixture. Our analysis shows that the dimer βLG-A does not change its size if the pH is varied from 5.60 to 4.58 and both species remain constant in size for temperature changes from 3.5° to 25°C Hydrodynamically, the octamers behave like closed-packed spheres with an effective radius of about 45 Å according to the Stokes-Einstein relation.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.     

Effects of estradiol administration in vivo on testosterone production in two populations of rat Leydig cells

Ricinus communis agglutinin, a galactose-binding lectin, agglutinates phospholipid/qlycolipid vesicles, phospholipid/glycolipid/cholesterol vesicles, low density lipoprotein, and the branched polysaccharide gum arabic. The temperature dependence of the initial velocity of agglutination is similar in all cases, with a maximum at ～25°C, and a decreased rate at lower and higher temperatures. The extent of agglutination is temperature-independent for T ≤ 25°C, and decreases with increasing temperature for T > 25°C. After agglutination at 25°C, an increase in temperature results in deagglutination. This effect is reversible upon return to 25°C. Thus RCA undergoes a reversible temperature-dependent transition between an agglutinating form (≤25°C) and a less active form (> 25°C). These observations have important consequences in the interpretation of experiments which attempt to correlate lectin-binding with temperature-dependent properties such as membrane fluidity or receptor lateral organization in natural and model membranes.     

“Setting paint” analogy for the hydrophobic self‐association of tropoelastin into elastin‐like hydrogel

Alkaline tropoelastin solutions (pH 11) were optically clear at low temperatures, but a firm gel formed when the temperature was raised to 37°C. Reversion to a clear solution took place if the temperature was lowered to below 20°C within less than 2 h, but not if 37°C was maintained for several hours. The precipitated elastin‐like hydrogel thus formed did not visually redissolve at low temperatures. Tropoelastin hydrogel was stable to subsequent washings with alkaline solution at 37°C, but at 4°C some hydrogel redissolved showing that association is at least partly reversible. Washing the hydrogel with neutral 8M urea solution at 4°C dissolved less than 10% of tropoelastin in 24 h. We characterized this phenomenon by combining temperature‐controlled light microscopy analysis, ¹H NMR spectroscopy (temperature, diffusion, and relaxation time studies), and UV‐absorption‐based concentration measurements. The self‐association of tropoelastin at pH 11 is due to hydrophobic interactions in an emulsion‐like system in which the spherules coalesce in a manner like a water‐based latex paint that forms a durable hydrophobic sheet as water and the organic solvent evaporate. In the present case, the sedimentation and entanglement of the tropoelastin porous sheets means that reverse dissolution is a kinetically slow process. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 321–330, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A Highlights from MBoC Selection: Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface.     

Investigations on the Thermal Stability of Fullerene-Based (Ag-C<Subscript>70</Subscript>) Nanocomposite Thin Films

Fullerene-based bi-functional nanocomposite thin film (Ag nanoparticles embedded in fullerene C₇₀ matrix) is synthesized by thermal co-deposition method. Thermal stability of Ag-C₇₀ nanocomposite is investigated by annealing the nanocomposite thin film at different temperatures from 80 to 350 °C for 30 min. Optical and structural properties of nanocomposite thin film with respect to high temperature are studied by UV-visible spectroscopy and x-ray diffraction, respectively. Transmission electron microscopy is performed to observe the temperature-dependent size evolution of Ag nanoparticles in fullerene C₇₀ matrix. A large growth of Ag nanoparticles is observed with temperature especially above 200 °C due to enhanced diffusion of Ag in fullerene C₇₀ at higher temperature and Ostwald ripening. The properties of metal-fullerene nanocomposite is not significantly affected up to a temperature of 150 °C. With a further increase in temperature, a major blue shift of ~?33 nm in SPR wavelength is seen at a temperature of 300 °C due to the thermal induced structural transformation of fullerene C₇₀ matrix into amorphous carbon. A very large-sized Ag nanoparticle with a wide size distribution varying from 27.8 ± 0.6 to 330.0 ± 4.5 nm is seen at 350 °C and due to which, a red shift of ~?16 nm is obtained at this temperature. This study throws light on the thermal stability of the devices based on metal-fullerene bi-functional nanocomposite.     

Seasonal patterns in the physiology of the European brown bear (Ursus arctos arctos) in Finland

The physiological indicators such as body temperature, blood chemistry and hematology of seven European brown bears (Ursus arctos arctos) were used in the present study. They were kept in either the Zoological Garden of University of Oulu (65°N, 25°24'E) or the Ranua Zoological Garden approx. 150 km NE of Oulu. Transmitters with a temperature-dependent pulse rate were implanted subcutaneously or into the abdominal cavity under anesthesia. Our data indicate that the body temperature of the bear decreases during the winter sleep to 4–5°C below the normal level (37.0–37.5°C). The lowest values, 33.1–33.3°C, were measured several times in midwinter. Hematocrit, hemoglobin and erythrocyte counts seem to be higher, and the leucocyte count lower during the denning period than in the awake bear. Plasma N-wastes were lower during the winter sleep than before or after it. The analysed blood parameters showed that plasma catecholamines and thyroid hormones decreased in the fall.     

Heat shock induces simultaneous rearrangements of all known cytoskeletal filaments in normal interphase fibroblasts

Hyperthermia (heat shock (HS)) induces changes in morphology of nucleoli, cytoplasmic organelles, and cytoskeleton. Responses to hyperthermia are, as a rule, similar in all types of eukaryote cells. However, there is no information on the uniformity of the cytoskeleton heat shock response (CHSR) in different cell types. This has led to the conclusion that the eukaryote CHSR depends on the cell type. We studied CHSR only in one cell type-in normal embryonic mouse fibroblasts (NEMFs) and in normal embryonic rat fibroblasts (NERFs), as well as in normal postnatal rat fibroblasts (NPRFs), by using the method of fluorescence microscopy. Incubation of the cells at 43°C led to a rearrangement of cytoskeleton. Responses of cytoskeleton to HS in NEMF, NERF, and NPRF were similar. Heat shock resulted in disassembly of bundles of actin filaments (AFs), marked changes in microtubule (MT) morphology, and collapse of intermediate filaments (IFs) around the nucleus. Rearrangements of different cytoskeleton filament types occurred simultaneously and were seen as soon as after 2–4 min. After 30–120 min of incubation at 43°C, the cells were still capable of rebuilding the actin cytoskeleton after the temperature had returned to normal (37°C). We believe that the cytoskeleton rearrangement under the action of HS can be of vital importance in cell protection against temperature stress. Data are discussed on possible coupling of the CHSR process with rearrangement of the protein synthesizing system, which leads to initiation and/or stimulation of synthesis of HS proteins.     

Temperature sensitivities of metazoan and pre-metazoan Src kinases

Homologous enzymes from different species display functional characteristics that correlate with the physiological and environmental temperatures encountered by the organisms. In this study, we have investigated the temperature sensitivity of the nonreceptor tyrosine kinase Src. We compared the temperature dependencies of c-Src and two Src kinases from single-celled eukaryotes, the choanoflagellate Monosiga brevicollis and the filasterean Capsaspora owczarzaki. Metazoan c-Src exhibits temperature sensitivity, with high activity at 30 °C and 37 °C. This sensitivity is driven by changes in substrate binding as well as maximal velocity, and it is dependent on the amino acid sequence surrounding tyrosine in the substrate. When tested with a peptide that displays temperature-dependent phosphorylation by c-Src, the enzymatic rates for the unicellular Src kinases show much less variation over the temperatures tested. The data demonstrate that unicellular Src kinases are temperature compensated relative to metazoan c-Src, consistent with an evolutionary adaptation to their environments.     

Diffusivity, viscosity, and cluster formation in protein solutions

The diffusion coefficient and viscosity of lysozyme solutions were measured at 25°C in various buffers with and without sodium chloride. Measurements were made over the entire concentration range in each case and were extended into the supersaturated region. The results show that diffusion coefficient behavior depends strongly on the buffer used and the ionic strength of the solution, which means the amount of sodium chloride used in buffer solution. Viscosity measurements indicate a small degree of time dependence, with the viscosity increasing with solution age.     

Binding of azide ion to methemoglobin at elevated temperatures and the reality of the "compensation" temperature

We have studied the binding of azide ion to ferrihemoglobin at elevated temperatures. Up to a temperature of 45°C there is no difference in the ligand binding behavior of hemoglobin when compared with the results obtained at lower temperatures. The compensation temperature Tc of 290.6 ± 5.3°K, obtained in this study within the temperature range 303–318°K, confirms that the compensation pattern obtained by Lumry and Rajender is not dependent on the temperature range of the experiment but an intrinsic property of the protein conformation.     

Temperature-dependent growth and emergence of functional leaves: an adaptive mechanism in the seedlings of the western Himalayan plant Podophyllum hexandrum

As an adaptive mechanism, hypocotyl dormancy delays emergence of functional leaf until favorable season of growth in Podophyllum hexandrum, an endangered medicinal plant of the western Himalayas. However, upon exposure of the freshly germinated seedlings to favorable temperature (25°C), functional leaves emerged within 20 days. Therefore, we examined regulation mechanisms of growth and development of this alpine plant by temperature under laboratory conditions. The seedlings were exposed to (1) 25°C (temperature prevailing at the time of maximum vegetative growth), (2) 4°C (mean temperature at the onset of winter in its natural habitat), and (3) 10°C (an intermediate temperature). Slackened growth at 4°C was followed by senescence of aerial parts and quiescence of roots and predetermined leaf primordia. Rapid development of leaf primordia at 25°C was associated with increased starch hydrolysis. This was evident from higher α-amylase activity and reducing sugars. These parameters decreased on sudden exposure to 4°C. In contrast, the roots (perennating organs) showed a slight increase (1.36-fold) in α-amylase activity. Growth and development in seedlings growing at 10°C (temperature less adverse than 4°C) were comparatively faster. The content of reducing sugars and α-amylase activity were also higher in all the seedling parts at 10°C as compared to 4°C. This indicated larger requirements for sugar by the seedlings at 10°C. Irrespective of temperature, maximum changes in nitrate and nitrate reductase occurred during the initial 10 days, i.e., when the readily available form of sugars (reducing sugar) was highest. This indicated that a temperature-dependent availability of carbon, but not temperature itself, was an important regulator of uptake and reduction of nitrogen. IHBT Publication number 508a.